Project 3 (Double-Strand Break Sensing, Signaling, and Repair) Integrates into SBDRS by focusing upon early DNA double strand break (DSB) repair and signaling responses. DSBs are highly toxic and mutagenic. Failure to expeditiously repair DSBs leads to cell death, chromosomal rearrangements, and human disorders, especially cancer. Proteins acting In DSB prevention and repair are associated with breast and ovarian cancer (BRCA1, BRCA2, Mre11), Nijmegen Breakage Syndrome (Nbs1, Rad50), and Ataxia telangiectasia (ATM, Mre11). DSB generation is moreover a primary means for cancer treatment; recent breakthroughs show that tumors with DSB repair deficiencies can be effectively targeted in combination therapies by poly-ADP ribose polymerase-1 inhibitors, a concept termed synthetic lethality. Thus, a molecular-level view of how cells sense, signal and repair DSBs is critical for understanding cancer risk, responses of normal and cancer cells to cancer treatments, and development of advanced cancer therapies that target the Achilles' heel of repair defects common to all cancer cells. Our project has led to seminal progress on structures, interactions, and conformational switches in the Mre11-Rad50-Nbs1 (MRN) complex and DNA-PK responses to DSBs. These results suggest that interaction states coupled with their allosteric regulation control biological outcomes to DSBs: they direct repair to either error-free or error-prone pathways, controlling genetic Integrity or aberration, and ultimately cell survival or death. Building upon our progress, we propose three Aims to characterize the structural cell biology of DSB responses for 1) the MRN complex, 2) DNA-PK and NHEJ complexes, and 3) DSB repair pathway coordination, control and regulation. Together these Alms will characterize pathway and cross-pathway interactions in vivo and In vitro for DSB response proteins to identify pathway connections and nodes for therapeutic intervention and synthetic lethality approaches. Project 3 has tight synergies with all other Projects and both scientific Cores. The anticipated outcome of the proposed cross-disciplinary experiments is a detailed molecular picture of the protein-DNA complexes, protein-protein interactions and functional states that orchestrate DSB sensing, repair, and signaling events mediated by MRN and DNA-PK. This picture will provide the molecular foundation for a detailed understanding of human diseases and cancer predispositions linked to MRN and DNA-PK proteins. Moreover, our analyses of interaction interfaces and conformational changes that control biological outcomes will highlight targets for therapeutic Interventions and provide chemical inhibitors as tools to test innovative new approaches for future advanced cancer therapies.